A critical pathological hallmark of Alzheimer?s disease (AD) is plaque deposits in the brain composed of aggregates of the small hydrophobic ?-amyloid (A?) peptide. It is well known that the 38-42 amino acid A? peptides at the crux of the prevalent ?Amyloid Hypothesis? of AD pathogenesis are generated by sequential proteolytic cleavages of the transmembrane ?-Amyloid precursor protein (APP). Two distinct proteolytic cascades lead to amyloidogenic versus non-amyloidogenic fates . Moreover, multiple pathogenic AD mutations alter the flux through these proteolytic pathways by unknown mechanisms. The lack of suitable assays to control or detect proteolysis of APP has both hindered the understanding of how proteolysis is regulated and the development of receptor specific modulators. We have recently developed a synthetic Notch proteolysis assay (SNAPS) that harnesses the easy to control inputs and outputs of Notch signaling to study proteolysis of diverse cell surface receptors by constructing receptor-of-interest/Notch chimeras. We will use this assay to study mechanism and find modulators of APP proteolysis. Aim 1: Determine how cellular environment, molecular environment and Alzheimer?s disease mutations affect APP proteolysis. Aim 2: Discover APP proteolytic modulators. We will identify nanobodies and single chain antibodies that bind to APP and potentially modulate its proteolysis into A? .